Radiofrequency device for increasing intracellular bioactive substance and plant cell culture method using the same

ABSTRACT

There are provided a radiofrequency device for increasing amount of a bioactive substance in a plant cell and a plant cell culture method for increasing amount of useful intracellular secondary metabolites by using the radiofrequency device. The cell culture method of the present invention makes it possible to increase specific secondary metabolites such as daidzein, equol, and the like in a cell and thus can be used for development into various medicines, agricultural pesticides, spices, pigments, food additives, and cosmetics containing bioactive substances. Further, the cell culture method of the present invention improves conventional cell culture methods limitedly used for specific cells or specific metabolites for increasing amount of intracellular bioactive substances and thus can be widely applied to production of cells and secondary metabolites.

FIELD OF THE INVENTION

The present invention relates to a radiofrequency device for increasingamount of a bioactive substance in a plant cell and a plant cell culturemethod for increasing amount of useful secondary metabolites in theplant cell by using the radiofrequency device.

BACKGROUND OF THE INVENTION

Plants are useful resources for producing a wide range of secondarymetabolites to be used for medicines, agricultural pesticides, spices,pigments, food additives, cosmetics, etc. However, if a secondarymetabolite is directly extracted from a plant, its content is low, andits productivity is highly varied depending on a cultivation conditionor a part of the plant, which causes an unstable supply of the secondarymetabolite. Further, while demands for secondary metabolites areincreased in food, medicine, and cosmetic fields, supplies of secondarymetabolites directly produced by cell culture are still limited. Inorder to solve such a problem, there have been various attempts tocontrol a mass production process and a culture condition in a bioreactor capable of mass-producing secondary metabolites.

Mass production of secondary metabolites through plant cell culture hasproblems of instability of a culture cell strain, low productivity, andslow growth, mass culture, and the like. In order to overcome theproblem of low productivity, conventionally, 1) manipulation ofnutriments of a culture medium such as addition of sugars, nitrates,phosphates, growth regulators, and precursors, or the like, 2)optimization of culture environment such as a culture temperature,lighting, acidity of a medium, etc., 3) treatment of derivatives forincreasing productivity, 4) cell membrane permeabilization and two-phaseculture for effectively collecting of secondary metabolites, and 5)modification of genes involving biosynthesis of secondary metabolitesand transfer of exotic genes have been used for increasing productivityof secondary metabolites. However, such methods can be limitedly usedfor specific cells or specific metabolites but cannot be generallyapplied to cell culture and secondary metabolites.

Meanwhile, a method for culturing cells is classified into a batchculture method in which a culture fluid and a cell are supplied once atthe beginning and there is no more supply or removal of nutriments, anda continuous culture method in which a culture fluid is put in anincubator and a new culture fluid is supplied from the outside at aconstant speed while the old culture fluid containing a cultured productis discharged to the outside at the same speed so as to continuouslyculture cells without lack of nutrients. The batch culture method is notsuitable for mass production. Therefore, as one of the continuousculture methods, a perfusion culture method in which a cell is left inan incubator and a medium containing a product is continuously collectedwhile a new medium is supplied has attracted attention. In this case, itis disclosed that cells and a medium are separated from each other byusing ultrasonic waves. To be specific, by using an ultrasonic cellseparator operated such that when cells start to be discharged to theoutside of the cell separator after ultrasonic waves are irradiated, theirradiation of the ultrasonic waves is stopped, and the cells arereturned to a bio reactor by applying a counter pressure with a pump,and then the ultrasonic waves are irradiated again, the cells and themedium are separated, whereby plant cells are cultured at a highdensity. However, in this case, the ultrasonic waves are used toseparate the cells and the medium, but cannot much contribute to anincrease in content of secondary metabolites of the cells. Further, ithas been pointed out that necrosis of the plant cells is often caused bythe ultrasonic waves.

-   Patent Document 1: Korean Patent Application No. 10-1997-0008119-   Patent Document 2: Korean Patent Application No. 10-2000-0017570

BRIEF SUMMARY OF THE INVENTION

An object of the present invention is to provide a cell culture methodfor increasing amount of a bioactive substance in a plant cell.

Further, another object of the present invention is to provide anoptimum condition of radiofrequency process intensity, a processingmethod, and the like for increasing amount of an intracellular bioactivesubstance.

Furthermore, still another object of the present invention is to providea radiofrequency device for increasing amount of an intracellularbioactive substance.

In order to achieve the above objects, an exemplary embodiment of thepresent invention provides a cell culture method for increasing amountof a bioactive substance in a plant cell, the method comprising: (a) aculturing step of inoculating the plant cell into a medium and culturingthe plant cell; and (b) a radiofrequency processing step of processingthe cultured cell with a radiofrequency for increasing amount of abioactive substance in the plant cell.

In an example of the present invention, the radiofrequency may be usedfor process in a range of 0.1 to 15 MHz.

In an example of the present invention, the culturing step may furthercomprise: (a1) a callus inducing step of culturing the plant cell in anMS medium containing IAA (indole acetic acid), BAP(6-benzylaminopurine), sucrose, and gelite for 2 to 5 weeks for inducinga callus from the germinated plant; and (a2) an adventitious rootinducing step of culturing the callus in an MS medium containing IBA(indole-3-butyric acid), MES monohydrate(2-(N-morpholino),benzyladenine, sucrose, and gelite for 2 to 5 weeks for inducing anadventitious root from the callus.

In an example of the present invention, the radiofrequency may be usedfor process 3 times every 2 to 10 minutes per day repeatedly for 5 to 15days.

In an example of the present invention, the bioactive substance may beat least one secondary metabolite selected from the group consisting ofalkaloids, flavonoids, terpenenoids, glycosides, and metabolitepigments.

In an example of the present invention, the flavonoid as a secondarymetabolite may be at least one metabolite selected from the groupconsisting of isoflavones, flavonols, flavanones, flavones,flavan-3-ols, and anthocyanins.

In an example of the present invention, the isoflavone may be at leastone secondary metabolite selected from the group consisting of daidzein,genistein, and equol.

Further, the present invention provides a radiofrequency cell incubatorcomprising: a bio reactor configured to culture a plant cellaccommodated therein by supplying a radiofrequency and air; aradiofrequency supplier configured to supply a radiofrequency to the bioreactor for increasing amount of an intracellular bioactive substance;an air supplier configured to supply air to the bio reactor; and anoscillograph connected to the radiofrequency supplier and configured torecord vibration of the bio reactor.

In an example of the present invention, the bio reactor may include acase in which an inlet opening for supplying a cell culture and aculture fluid is provided at an upper part, radiofrequency installationunits are aligned at both sides in the same line, and an air inletopening is provided at a lower part; an upper cover configured to openor close the inlet opening; radiofrequency terminals installed at therespective radiofrequency installation units and configured to transmita radiofrequency transmitted from the radiofrequency supplier; ahorizontal supporting plate formed in a plate shape and including fixingholes for supporting the case in the center; and fixing rods verticallyinstalled under the horizontal supporting plate along a periphery of thehorizontal supporting plate.

In an example of the present invention, buffer members may be installedat connection portions between the case and the horizontal supportingplate and between the horizontal supporting plate and the fixing rods,respectively, to reduce shock and vibration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic diagram showing a method for inducing callusesfrom a plant.

FIG. 1B provides photos showing induced calluses according to adesirable example of the present invention. (duplicate experiments)

FIG. 2 shows an HPLC result obtained by identifying a substanceincreased during a radiofrequency process onto a plant cell.

FIG. 3 is a graph showing that daidzein is increased during aradiofrequency process onto a plant cell.

FIG. 4 is a graph showing that equol is increased during aradiofrequency process onto a plant cell.

FIG. 5 is a configuration view showing a radiofrequency cell incubatoraccording to the present invention.

FIG. 6 is a cross-sectional view showing a bio reactor constituting aradiofrequency cell incubator according to the present invention.

FIG. 7 is a reference photo showing an actual image of a radiofrequencycell incubator according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a radiofrequency device for increasingamount of an intracellular bioactive substance and a cell culture methodfor increasing amount of useful secondary metabolites in a cell by usingthe radiofrequency device.

Secondary metabolites refer to natural products that do not have primaryand direct physiological functions, such as growth, development, andreproduction of an organism, required for survival and are limitedlyproduced by specific organisms.

The secondary metabolites are mainly produced by plants andmicroorganisms, and it is not too much to say that most of major anduseful metabolites are derived from plants and microorganisms. Unlikeplants, animals are different in terms of genetic composition, nutritionsupply source, and storage type, and they have mobility and thus caneasily obtain food required for daily life and also can avoid dangerthreatening life. Meanwhile, in the case of plants without mobility,they protect themselves by producing toxic substances as a measure ofself-defense and generate secondary metabolites as a means to lureinsects for nutrition competition or propagation of species.

Conventionally, a technology for analyzing and extracting such secondarymetabolites was not developed and plants containing useful substanceswere searched by using five senses. However, recently, along withdevelopment of natural product chemistry, biology, pharmacology, andbiochemistry, analysis methods have been developed, so that it ispossible to accurately extract a trace of a substance and also possibleto determine a structure thereof. Along with this trend, there has beenconducted research focusing on a method for producing more varioussecondary metabolites in more amounts based on usability of secondarymetabolites.

Synthesis and accumulation of secondary metabolites may be often carriedout limitedly at a specific time in specific organs, tissues, or cells.Thus, it is important to catch such an appropriate time and extractsufficient secondary metabolites. Currently, production of secondarymetabolites depends largely on cell culture. This is because cellculture exhibits a high growth rate as compared with an organism and isnot influenced by local and climatic restrictions and less damaged bydisease and insects through sterile culture, whereby it is possible tocarry out uniform culture regardless of environmental conditions.Further, cell culture can be carried out under an environmentartificially regulated to promote production of specific secondarymetabolites.

Secondary metabolites can be classified into four kinds, i.e. alkaloids,phenol compounds, terpenes, and other metabolites, depending on astructure and a synthesis process.

Alkaloid is an aromatic nitrogen compound and has a heterocyclicstructure including a nitrogen atom. A molecule of the alkaloid isalkaline due to the positively charged nitrogen atom and generallywater-soluble. The alkaloids can be classified into several kinds, i.e.pyrrolidine, tropane, piperidine, pyrrolizidine, quinolizidine,isoquinoline, indole, etc., depending on a structure of a heterocycle.There have been found about 100,000 or more kinds of alkaloidsaccounting for 20 to 30% of vascular plants and mostly distributed inplants. Most of them are found from herbaceous dicotyledones, and it isknown that a plant such as poppy or cinchona contains 20 or more kindsof alkaloids. Although functions of alkaloids in plants are not known,the alkaloids have attracted lots of attention due to their strongphysiological effects on animals including mammals. Nicotine, caffeine,cocaine, and the like have a function of a stimulant or a sedative andthus have been widely used as favorite foods. Morphine, codeine,atropine, ephedrine, quinine, and the like have been usefully used asmedicines.

Meanwhile, a phenol compound is a generic term for secondary metaboliteshaving an aromatic cyclic structure including a substitutable hydroxylgroup. A phenol compound of a plant includes chemically heterogeneoussubstances some of which are dissolved in an organic solvent only andsome of which are water-soluble carboxyl acids, glycosides, and macropolymers. A function of the phenol compound is as varied as a structurethereof and includes a defense function against attack of an herbivoreor a pathogen, a mechanical supporting function, a function of inducingpollination vectors and dispersing seeds, a function of inhibitinggrowth and development of adjacent competitor plants.

The phenol compounds include flavonoids. There are many kinds offlavonoids which can be classified into four kinds, i.e. anthocyanins,flavones, flavonoles, and isoflavones, depending on a structure and aposition of a substituent.

The anthocyanins are pigments found in red, pale pink, purple, and bluefruit and flowers and capable of luring animals to spread pollen andseeds, and it is known that the anthocyanins can improve functions ofeyes and liver.

The flavones and flavonoles can be found in flowers and generally absorblight having a shorter wavelength as compared with the anthocyanins.Therefore, these pigments are invisible to the human eye but visible toinsects such as bees capable of seeing ultraviolet rays.

The isoflavones have structural similarities and functional similaritiesto estrogens and have effects of the estrogens in the body as one ofphytoestrogens. Therefore, it is reported that the isoflavones have theeffect of preventing osteoporosis, cancer, cardiovascular diseases, andthe like. If the estrogens are used for a long time, they may cause sideeffects of a risk of developing cancer, headache, and the like, which isnow being debated. Meanwhile, the isoflavones act as agonists orantagonists depending on a bonding ability of an estrogen receptor butside effects thereof have rarely been reported. The Food and DrugAdministration recommends intake of 25 mg or more of isoflavones perday.

Daidzein is one of isoflavone compounds separated from a soybean. It isknown that the daidzein works to lower blood cholesterol and thus it iseffective in preventing and treating for various chronic diseases. Thedaidzein has a structure similar to that of estrogen, and, thus, aninternal absorption rate thereof is high. The daidzein is activated bybeing bonded to an estrogen receptor. As compared with estrogen therapycausing side effects such as breast cancer, the daidzein has no sideeffect, and, thus, it has attracted attention as an alternativesubstance to the estrogen. It is known as a highly effective antioxidantcapable of reducing damage caused by free radicals in tissue, and itserves as a dietary supplement formulation effective for menopausalwomen through a function similar to that of the estrogen. Further,regarding cancer cell growth in the body, the daidzein can be used as amaterial for controlling random growth of cells and appropriatelyregulating cell growth and cell division.

Meanwhile, equol is known as a metabolite of the daidzein. It isreported that the equol inhibits DHT (dihydrotestosterone) as a malehormone causing prostate cancer and male pattern baldness. That is, itwas confirmed that when the equol was injected into a male mouse,prostate became small. Further, when DHT was injected into a mouseincapable of generating the DHT due to removal of its testicles,prostate of the mouse became large, but it was confirmed that in thecase of injecting the equol together, such a change was not observed.

Further, the terpenes have the greatest diversity among secondarymetabolites and are known as having an insecticidal effect. Moreover,other secondary metabolites such as glycosides, glucosinolates,canavanines, etc. have been known.

The present inventors conducted research on plant cells for culturingcells containing high levels of useful secondary metabolites.

Above all, the present inventors induced a callus by using a soybeanplacenta for culturing cells derived from plants and induced anadventitious root from the callus. The term “callus” means tissue formedwhen a plant body is wounded and tissue formed at the wounded part ofthe plant body, and mainly refers to amorphous tissue or mass of cellsformed by culturing segments, organs, tissues, or the like and losingability to cause of normal organogenesis or histodifferention. Thecallus is mostly comprised of parenchyma cells and can be a common namefor plant tumor tissues formed by infection of agrobacterium or the likein a broad sense

Nowadays, the callus refers to specific tissue or mass of cells formedwhen tissue cut off from a plant body is cultured in a medium containingauxin, when a certain kind of a plant is wounded, or when a wounded partis treated with auxin. The callus is undifferentiated plastid and can bedifferentiated into various organs such as stems, roots, and leaves byvarious stimuli. Thus, it is sometimes referred to as a stem cell.

The callus becomes a useful means for tissue culture. The term “tissueculture” means a technology in which a callus is formed by culturing anorgan, a tissue, or a cell separated from a plant in an artificialmedium containing nutrients to maintain, differentiate, and proliferatea plant body. Unlike an animal cell, a plant cell has a totipotency(ability of a cell or tissue to form a shape of the whole cell or toreproduce a plant body and to be differentiated into all of cells) as anability to be differentiated into all of cells, which enables suchtissue culture.

According to plant tissue culture, an organ, a tissue, or a cell of aplant comprised of lots of cells is extracted and separated from a plantbody and cultured in a reactor containing nutrients to induce a callusor a group of single cells or to redifferentiate the plant body or organinto a plant body in the callus or the cell.

A callus can be generated at any one part of leaves, stems, roots, etc.through such tissue culture, and a callus can be continuously generatedby subculture. Further, by repeating proliferation of a completelyundifferentiated cell and regulating an amount of a plant hormone in amedium, an irregular vessel or phloem can be differentiated, anadventitious bud or an adventitious root can be formed, and an intactindividual can be formed at the callus. Therefore, the callus is a keymaterial for research on formation of a shape.

The present inventors generated a callus at a soybean placenta andinduced an adventitious root from the callus and then tissue-culturedthe adventitious root by using a radiofrequency cell incubator 100. Thatis, the present inventors tried to increase secondary metabolites asuseful intracellular substances by carrying out a tissue culture stepwith a radiofrequency.

The tissue culture can be carried out in a bio reactor of theradiofrequency cell incubator 100. The bio reactor refers to anapparatus used for producing substances by using a characteristic of anenzyme capable of effectively regulating a high specificity reaction atnormal temperature and pressure. Although the bio reactor originallyrefers to an apparatus for culturing a microorganism or a cell toproduce substances by using a reaction of living body, it often refersto a reactor allowing various chemical reactions to be carried out byusing an immobilized enzyme or a microorganism and has been used tomass-produce useful substances on an industrial scale or to diagnose adisease through microdetermination of a specific biogenic substance.Unlike a conventional culture method in which a solid medium prepared isexplanted in each bottle, in the case of the bio reactor, a large amountof a liquid medium is put into the reactor and cultured while air issupplied into the reactor. Therefore, with the bio reactor, as comparedwith the conventional culture method, culture can be carried out in alarge amount at the same time along with excellent growth, and, thus,excellent shoots can be obtained in a much shorter time. Further, thebio reactor has been used to extract main substances from a plant. Inthe present invention, there has been used a bio reactor equipped with aradiofrequency supplier for particularly increasing amount ofintracellular secondary metabolites, and a configuration thereof will bedescribed in detail below.

The radiofrequency cell incubator 100 as the present invention iscomprised of a bio reactor 200, a radiofrequency supplier configured tosupply a radiofrequency to the bio reactor 200, an air supplier 400configured to supply air to the bio reactor 200, and an oscillograph 500connected to the radiofrequency supplier 300 and configured to recordvibration of the bio reactor 200.

The bio reactor 200 is configured to culture a cell culture accommodatedtherein by supplying a radiofrequency and air thereto.

The bio reactor 200 carried out as described above is comprised of acase 210 having a predetermined size, an upper cover 220 detachablyprovided at an upper part of the case 210, radiofrequency terminals 230aligned at both sides of the case 210 in the same line, a horizontalsupporting plate 240 supporting the case 210, and fixing rods 250 havinga predetermined height and supporting the horizontal supporting plate240.

Further, in the case 210, an inlet opening 211 for supplying a cellculture and a culture fluid is provided at an upper part, radiofrequencyinstallation units 212 are aligned at both sides in the same line, andan air inlet opening 213 is provided at a lower part.

That is, the case 210 is formed in an upside-down gourd bottle shape,and the inside thereof communicates with the inlet opening 211, theradiofrequency installation units 212, and the air inlet opening 213provided at the upper part, the both sides in the same line, and thelower part, respectively.

Furthermore, the upper cover 220 detachably provided at the upper partof the case 210 opens or closes the inside of the case 210 by locking orunlocking.

Then, the radiofrequency terminals 230 respectively provided at theradiofrequency installation units 212 constituting the case 210 transmita radiofrequency transmitted from the radiofrequency supplier 300 to theinside of the case 210.

Further, in the present invention, the radiofrequency terminal 230 iscomprised of a terminal portion 231 formed in “┤” shape, a sealingmember 232 provided at one side of the terminal portion 231 and tight onthe radiofrequency installation unit 212 of the case 210, and aconnection line 233 of which one side is connected to the terminalportion 231 and the other side is connected to the radiofrequencysupplier 300.

That is, the radiofrequency terminal 230 is configured to receive aradiofrequency through the connection line 233 connected to theradiofrequency supplier 300 and propagate the radiofrequency into thecase 210 thorough the terminal portion 231.

Furthermore, the horizontal supporting plate 240 supporting the case 210is manufactured into a plate having predetermined thickness and diameterand includes fixing holes 241 for fixing the case 210 in the center.

Moreover, fixing rods 250 vertically installed under the horizontalsupporting plate 240 along a periphery of the horizontal supportingplate 240 may be formed of one selected from publicly known rods or barshaving a predetermined height.

In this case, buffer members 260 made of a material selected fromrubber, silicon, and synthetic resin are installed at connectionportions between the case 210 and the horizontal supporting plate 240and between the horizontal supporting plate 240 and the fixing rods 250,respectively, to reduce shock and vibration.

Herein, the buffer member 260 is formed in a hollow shape and mayselectively include embossing projections at intervals at its upper andlower parts.

The radiofrequency supplier 300 comprised of an operation unit (notillustrated) configured to generate a radiofrequency, a manipulationunit 310 configured to regulate an on/off operation and aradiofrequency, a display unit 320 configured to display a currentstatus and an operation status, and a level meter 330 configured todisplay a status of a frequency supplies a radiofrequency to the bioreactor 200.

Herein, the operation unit is formed of one selected from operationunits constituting publicly known radiofrequency suppliers, and detailedexplanation thereof will be omitted.

That is, the radiofrequency supplier 300 supplies a radiofrequency tothe radiofrequency terminals 230 of the bio reactor 200 while theoperation unit is operated in association with an operation of themanipulation unit 310, and a current status can be monitored in realtime through the display unit 320 and the level meter 330.

The air supplier 400 has a predetermined size and includes an airgeneration unit (not illustrated) therein. Discharge openings 420configured to discharge air are formed in front, and an air hose 440configured to deliver air is installed at the discharge opening 420 soas to supply air to the bio reactor 200.

Herein, the air generation unit is formed of one selected from airgeneration units constituting publicly known air suppliers or airsupplying devices, and detailed explanation thereof will be omitted.

That is, the air generation unit 400 generates air through the airgeneration unit in response to an input signal and supplies the air tothe case 210 of the bio reactor 200 through the discharge opening 420and the air hose 440.

The oscillograph 500 is comprised of a vibration operation unit (notillustrated) configured to convert and record vibration, a vibrationmanipulation unit 510 configured to regulate an on/off operation andvarious functions, a vibration display unit 520 configured to display acurrent status, and a connection line 530 connected to theradiofrequency supplier 300.

Herein, the vibration operation unit is formed of one selected fromvibration operation units installed at publicly known oscillographs, anddetailed explanation thereof will be omitted.

That is, the oscillograph 500 enables real-time monitoring of avibration status of the bio reactor 200 through the connection line 530connected to the radiofrequency supplier 300 and the vibration displayunit 520.

An example of the radiofrequency cell incubator 100 configured asdescribed above will be explained below.

Above all, the a case 210 having a predetermined size, the upper cover220 detachably provided at an upper part of the case 210, theradiofrequency terminals 230 aligned at both sides of the case 210 inthe same line, the horizontal supporting plate 240 supporting the case210, and the fixing rods 250 having a predetermined height andsupporting the horizontal supporting plate 240 are prepared.

Then, the radiofrequency supplier 300 comprised of the operation unit(not illustrated) configured to generate a radiofrequency, themanipulation unit 310 configured to regulate an on/off operation and aradiofrequency, the display unit 320 configured to display a currentstatus and an operation status, and the level meter 330 configured todisplay a status of a frequency is installed at an interval with the bioreactor 200.

Thereafter, the air supplier 400 having a predetermined size andincluding an air generation unit (not illustrated) therein, thedischarge openings 420 configured to discharge air and formed in frontthereof, and the air hose 440 configured to deliver air and installed atthe discharge opening 420 is installed at an interval with theradiofrequency supplier 300.

Then, when the oscillograph 500 comprised of the vibration operationunit (not illustrated) configured to convert and record vibration, thevibration manipulation unit 510 configured to regulate an on/offoperation and various functions, the vibration display unit 520configured to display a current status, and the connection line 530connected to the radiofrequency supplier 300 is installed at one side ofthe air supplier 400, assembly of the radiofrequency cell incubator 100is completed.

Herein, an assembly sequence of the radiofrequency cell incubator 100may be different from the above-described sequence.

Hereinafter, a use state of the radiofrequency cell incubator 100configured as described above will be explained below.

Above all, the upper cover 220 closing the inlet opening 211 of the case210 constituting the bio reactor 200 is opened. Then, a culture fluidand a cell culture are supplied into the case 210 and the inlet opening211 is closed by using the upper cover 220.

Further, a set value of a radiofrequency is regulated by using themanipulation unit 310 constituting the radiofrequency supplier 300.Then, the radiofrequency is supplied to the radiofrequency terminals 230of the bio reactor 200 through the connection line 233.

At the same time, air generated by the air supplier 400 is supplied tothe air inlet opening 213 of the bio reactor 200 through the air hose440.

Thereafter, the cell culture accommodated within the case 210 isinfluenced by the radiofrequency transmitted through the radiofrequencyterminals 230, and a user can monitor radiofrequency informationsupplied in real time through the display unit 320 and the level meter330.

Furthermore, vibration of the bio reactor 200 transmitted to theradiofrequency supplier 300 is recorded by the oscillograph 500.

Hereinafter, there will be explained Examples of a cell culture methodfor increasing amount of bioactive substances by using theradiofrequency device. These Examples are provided for the purpose ofillustration, but the scope of the present invention cannot be limitedthereto.

Example 1 Surface Disinfection of Cultured Tissue

The present inventors used a soybean placenta to culture a plant cellbased on the idea that placenta tissue is tissue for forming seeds of aplant and various phytochemicals are expressed and a level of suchexpression is high. The placenta refers to tissue where an ovule islocated in an ovary of a pistil, and the placenta is usually positionedat the edge of an carpel constituting the ovary and sometimes positionedat a medius of the carpel and may be formed in a straight pillar shapeat a base of the ovary or from the base in the ovary.

The present inventors first carried out a disinfection process in orderto induce a callus from the soybean placenta tissue. To do so, thesoybean placenta was cut into appropriate-sized pieces, and a surface ofthe placenta was washed with running water for about 6 hours. Thesoybean placenta tissue clearly washed was immersed in 70% ethanol for 5seconds for primary disinfection and then washed with sterile water 3times and treated in a 4% NaOCl solution for 10 minutes for surfacesterilization. Then, the soybean placenta tissue was washed with sterilewater 3 times in a clean bench and moisture thereof was removed withsterile filter paper.

Example 2 Induction of Callus

The present inventors cut the washed soybean placenta tissue into piecesabout 5 mm long about 5 mm wide. In order to induce calluses therefrom,the washed plant tissue was explanted and cultured in a MS (Murashigeand Skoog) medium containing 2 mg/L of IAA (indole acetic acid) as oneof auxins as plant growth regulators, 2 mg/L of BAP(6-benzylaminopurine) as one of cytokinins, 30 g/L of sucrose, and 2 g/Lof gelite as an agar medium at 25±2° C. for 4 weeks. As a result, as canbe seen from FIG. 1B, a callus was observed.

Example 3 Induction of Adventitious Root

The present inventors induced an adventitious root by using the callusinduced in Example 2. An adventitious root refers to a root which is notdeveloped from an apical meristem derived from a seed but shown duringcutting of a tree, cutting of a leaf, or tissue culture.

In order to culture the adventitious root, the present inventorsexplanted and cultured the callus in a MS (Murashige and Skoog) mediumcontaining 2 mg/L of IBA (indole-3-butyric acid) as an plant growthregulator, 0.5 g/L of MES Monohydrate(2-(N-morpholino) ethanesulfonicacid)), 2 mg/L of benzyladenine, 30 g/L of sucrose, and 2 g/L of geliteas an agar medium at 21±1° C. for 4 weeks. The culture was carried outat a pH of 5.8 in a dark room condition.

While culturing the adventitious root, the present inventors made itpossible to produce a substance from which root initiation wasinevitable for expression of biosynthesis.

Example 4 Comparison of Bioactive Substances Induced by RadiofrequencyProcess

The present inventors tried to compare bioactive substances induced byperforming a radiofrequency process onto the induced adventitious root.Above all, while culturing the adventitious root in the radiofrequencycell incubator, the present inventors applied a radiofrequency of 240kHz or 270 kHz to the adventitious root 3 times every 5 minutes for aday and repeated this process for a week or two weeks. A reaction volumeof a bio reactor was 1 L, power was 20 W, and a cell culture mediumcontained 4.4 g of MS medium powder, 30 g of sucrose, 2 mg of benzyladenine, 2 mg of IAA, and MES (pH 5.8) for buffering.

The sample cultured in the medium for a week with a radiofrequencyprocess was collected and vacuum-dried for 6 to 10 hours. Then, thesample was ultrasonic extracted with 50% ethanol as a solvent for 1 hourand vortexed for 30 minutes. When secondary metabolites weresufficiently released to a supernatant, the supernatant was obtained bycentrifugation. All samples were analyzed by using an HPLC (HPLC (Waters650E Advanced Protein Purification System) and Gemini 5u C18 110A(4.6×250 mm, 5 micron, Phenomenex Co., Ltd.) was used as a column. Whilethe analysis was carried out, a concentration gradient of water(containing 0.1% TFA (Trifluoro acetic acid)): acetonitrile (containing0.1% TFA (Trifluoro acetic acid)) in a mobile phase solvent was changedfrom 10:0 in initial 0 to 45 minutes to 1:9 in a time range of 45 to 50minutes and a flow rate was 1 ml/min A UV detector (230 nm) was used asa detector.

The amount of the secondary metabolites was remarkably increased by theradiofrequency process as compared with a case where the radiofrequencyprocess was not carried out, and a profile of the secondary metaboliteswas analyzed by the HPLC. As a result of the analysis, it could be seenthat daidzein and equol showed most remarkable results (FIG. 2). Thatis, it was observed that the amount of the daidzein was increased toabout 5.4 times by the radiofrequency process. Further, it was observedthat when the radiofrequency process was carried out with aradiofrequency of 240 kHz and power of 20 W 3 times every 5 minutes fora day repeatedly for two weeks, the amount of the equol as one ofisoflavones was also increased to about 5.9 times (FIG. 4).

The present invention makes it possible to increase intracellularbioactive substances in various plant cells and thus can be used fordevelopment into various medicines, agricultural pesticides, spices,pigments, food additives, and cosmetics containing bioactive substances.Further, the present invention improves the conventional cell culturemethods limitedly used for specific cells or specific metabolites forincreasing amount of intracellular bioactive substances and thus can bewidely applied to production of cells and secondary metabolites.

The present invention has been shown and described with reference tocertain exemplary embodiments thereof. It will be understood by anordinary person skilled in the art that the present invention can bemodified and changed in form without departing from the spirit and scopeof the present invention. Therefore, it should be considered that thedisclosed exemplary embodiments are not intended to limit the presentinvention but intended to illustrate the present invention. The scope ofthe present invention is defined not by the above description but theappended claims, and all differences within the scope equivalent theretoshould be construed as being included in the present invention.

What is claimed is:
 1. A cell culture method for increasing amount of anintracellular bioactive substance, the method comprising: (a) aculturing step of inoculating a plant cell into a medium and culturingthe plant cell; and (b) a radiofrequency processing step of processingthe cultured cell with a radiofrequency for increasing amount of anintracellular bioactive substance in the plant cell.
 2. The cell culturemethod of claim 1, wherein the radiofrequency is used for process in arange of 0.1 to 15 MHz.
 3. The cell culture method of claim 2, whereinthe culturing step further includes: (a1) a callus inducing step ofculturing the plant cell in an MS (Murashige and Skoog) mediumcontaining IAA (indole acetic acid), BAP (6-benzylaminopurine), sucrose,and gelite for 2 to 5 weeks for inducing a callus from a germinatedplant; and (a2) an adventitious root inducing step of culturing thecallus in an MS medium containing IBA (indole-3-butyric acid), MESmonohydrate(2-(N-morpholino), benzyladenine, sucrose, and gelite for 2to 5 weeks for inducing an adventitious root from the callus.
 4. Thecell culture method of claim 3, wherein the radiofrequency is used forprocess 3 times every 2 to 10 minutes per day repeatedly for 5 to 15days.
 5. The cell culture method of claim 1, wherein the bioactivesubstance is at least one secondary metabolite selected from the groupconsisting of alkaloids, flavonoids, terpenenoids, glycosides, andmetabolite pigments.
 6. The cell culture method of claim 5, wherein theflavonoid as a secondary metabolite is at least one secondary metaboliteselected from the group consisting of isoflavones, flavonols,flavanones, flavones, flavan-3-ols, and anthocyanins.
 7. The cellculture method of claim 6, wherein the isoflavone is at least onesecondary metabolite selected from the group consisting of daidzein,genistein, and equol.
 8. A radiofrequency cell incubator comprising: abio reactor configured to culture a plant cell accommodated therein bysupplying a radiofrequency and air; a radiofrequency supplier configuredto supply a radiofrequency to the bio reactor for increasing amount ofan intracellular bioactive substance; an air supplier configured tosupply air to the bio reactor; and an oscillograph connected to theradiofrequency supplier and configured to record vibration of the bioreactor.
 9. The radiofrequency cell incubator of claim 8, wherein thebio reactor includes a case in which an inlet opening for supplying acell culture and a culture fluid is provided at an upper part,radiofrequency installation units are aligned at both sides in the sameline, and an air inlet opening is provided at a lower part; an uppercover configured to open or close the inlet opening; radiofrequencyterminals installed at the respective radiofrequency installation unitsand configured to transmit a radiofrequency transmitted from theradiofrequency supplier; a horizontal supporting plate formed in a plateshape and including fixing holes for supporting the case in the center;and fixing rods vertically installed under the horizontal supportingplate along a periphery of the horizontal supporting plate.
 10. Theradiofrequency cell incubator of claim 9, wherein buffer members areinstalled at connection portions between the case and the horizontalsupporting plate and between the horizontal supporting plate and thefixing rods, respectively, to reduce shock and vibration.